/ . Biochem. 83, 1533-1543 (1978)
Studies on Pig Serum Lipoproteins Optical Properties of Low Density Lipoproteins Jun-ichi AZUMA,* Naoki KASHIMURA,* and Tohru KOMANO* •Biochemical Laboratory, Department of Agricultural Chemistry, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, December 16, 1977
The circular dichroism (CD), optical rotatory dispersion (ORD), and fluorescence emission spectra of two subfractions of pig serum low density lipoproteins (LDL! and LDL,) were compared. The contribution of the carbohydrate moiety to the CD and ORD spectra was estimated on the basis of data obtained from isolated glycopeptides and the constituent monosaccharides. The carbohydrate moiety had no effect on the conformation of the protein moieties of LDLj and LDL, (apoLDLj and apoLDL,). However, the intensities of the observed extrema in the CD and ORD spectra of the glycopeptides were greater than those expected from the monosaccharide composition. This suggests the existence of secondary structure in the carbohydrate moiety. In contrast to the carbohydrate moiety, the contribution of the lipid moiety to the CD and ORD spectra could not be neglected. When the effect of the lipid moiety was subtracted from the CD and ORD spectra, the spectra due to apoLDLj and apoLDL, were quite similar. Delipidation in the presence of sodium dodecyl sulfate (SDS) induced an increase in the content of disordered structure and a-helix accompanied by a decrease in the ^-structure. In the presence of SDS, marked quenching occurred in the fluorescence emission spectra with a blue shift of the maximum emission wavelength from 332 to 326 nm. ApoLDLj and apoLDLj showed quite similar SDS-induced conformational transitions. The secondary structures of apoLDLi and apoLDL, in the native lipoproteins were stable to changes of pH and temperature. However, this stability was lost in the presence of SDS. These results suggest the importance of the lipid moiety in maintaining the native secondary structures of LDL^ and LDL,. From the overall similarity of the optical properties of apoLDLj and apoLDL,, we conclude that the secondary structures of apoLDLi and apoLDL, are identical.
Abbreviations: LDL, total low density lipoprotein of density 1.007-1.090 g/ml; LDLt and LDL,, subfractions of LDL having hydrated densities of 1.033 and 1.050 g/ml, respectively; apoLDL, apoLDLi and apoLDL,, lipid-free protein from total LDL, LDL, and LDL,, respectively; SDS, sodium dodecyl sulfate; Man, D-mannose; Gal, D-galactose; Fuc, L-fucose; NANA, 5-acetamido-3,5-dideoxy-D-^/yc?ro-r>^a/oc/o-nonulosonic acid; GlcNAc, 2-acetamido-2-deoxy-D-glucose; Me a-GlcNAc, methyl 2-acetamido-2-deoxy-a-r>glucopyranoside; Me /9-GlcNAc, methyl 2-acetamido-2-deoxy-/J-r>glucopyranoside. Vol. 83, No. 6, 1978
1533
Downloaded from https://academic.oup.com/jb/article-abstract/83/6/1533/2185775 by Serials Dept user on 11 January 2019
V.
J. AZUMA, N. KASHIMURA, and T. KOMANO
1534
Circular dichroism (CD) and optical rotatory dispersion (ORD) have been widely used for the determination of the secondary structures of proteins (7-77). However, in determining the conformation of apoLDL in native LDL, it is important to clarify the contribution of the lipid and carbohydrate moieties to the CD and ORD spectra. The technique of fluorescence emission spectroscopy can be used to overcome this problem since the fluorescence of proteins specifically originates from the aromatic amino acids (72). The aim of the present work was to clarify the conformations of apoLDLj and apoLDL, by the following procedure: 1) measurement of the CD and ORD spectra of native LDLj and LDL,; 2) evaluation of the contributions of the carbohydrate and lipid moieties to the CD and ORD spectra; 3) determination of the CD and ORD spectra due to the protein moiety by subtracting the contributions of the lipid and carbohydrate moieties; 4) comparison of the corrected results for secondary structures of apoLDLi and apoLDL, in native LDLj and LDL, with those obtained in the presence of SDS; 5) determination of the pH- and temperature-dependent conformational changes; and 6) comparison of the results obtained from the CD spectra with those from fluorescence emission spectra. MATERIALS AND METHODS Materials—Total low density lipoprotein (LDL,
Studies on Pig Serum Lipoproteins Optical Properties of Low Density Lipoproteins Jun-ichi AZUMA,* Naoki KASHIMURA,* and Tohru KOMANO* •Biochemical Laboratory, Department of Agricultural Chemistry, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, December 16, 1977
The circular dichroism (CD), optical rotatory dispersion (ORD), and fluorescence emission spectra of two subfractions of pig serum low density lipoproteins (LDL! and LDL,) were compared. The contribution of the carbohydrate moiety to the CD and ORD spectra was estimated on the basis of data obtained from isolated glycopeptides and the constituent monosaccharides. The carbohydrate moiety had no effect on the conformation of the protein moieties of LDLj and LDL, (apoLDLj and apoLDL,). However, the intensities of the observed extrema in the CD and ORD spectra of the glycopeptides were greater than those expected from the monosaccharide composition. This suggests the existence of secondary structure in the carbohydrate moiety. In contrast to the carbohydrate moiety, the contribution of the lipid moiety to the CD and ORD spectra could not be neglected. When the effect of the lipid moiety was subtracted from the CD and ORD spectra, the spectra due to apoLDLj and apoLDL, were quite similar. Delipidation in the presence of sodium dodecyl sulfate (SDS) induced an increase in the content of disordered structure and a-helix accompanied by a decrease in the ^-structure. In the presence of SDS, marked quenching occurred in the fluorescence emission spectra with a blue shift of the maximum emission wavelength from 332 to 326 nm. ApoLDLj and apoLDLj showed quite similar SDS-induced conformational transitions. The secondary structures of apoLDLi and apoLDL, in the native lipoproteins were stable to changes of pH and temperature. However, this stability was lost in the presence of SDS. These results suggest the importance of the lipid moiety in maintaining the native secondary structures of LDL^ and LDL,. From the overall similarity of the optical properties of apoLDLj and apoLDL,, we conclude that the secondary structures of apoLDLi and apoLDL, are identical.
Abbreviations: LDL, total low density lipoprotein of density 1.007-1.090 g/ml; LDLt and LDL,, subfractions of LDL having hydrated densities of 1.033 and 1.050 g/ml, respectively; apoLDL, apoLDLi and apoLDL,, lipid-free protein from total LDL, LDL, and LDL,, respectively; SDS, sodium dodecyl sulfate; Man, D-mannose; Gal, D-galactose; Fuc, L-fucose; NANA, 5-acetamido-3,5-dideoxy-D-^/yc?ro-r>^a/oc/o-nonulosonic acid; GlcNAc, 2-acetamido-2-deoxy-D-glucose; Me a-GlcNAc, methyl 2-acetamido-2-deoxy-a-r>glucopyranoside; Me /9-GlcNAc, methyl 2-acetamido-2-deoxy-/J-r>glucopyranoside. Vol. 83, No. 6, 1978
1533
Downloaded from https://academic.oup.com/jb/article-abstract/83/6/1533/2185775 by Serials Dept user on 11 January 2019
V.
J. AZUMA, N. KASHIMURA, and T. KOMANO
1534
Circular dichroism (CD) and optical rotatory dispersion (ORD) have been widely used for the determination of the secondary structures of proteins (7-77). However, in determining the conformation of apoLDL in native LDL, it is important to clarify the contribution of the lipid and carbohydrate moieties to the CD and ORD spectra. The technique of fluorescence emission spectroscopy can be used to overcome this problem since the fluorescence of proteins specifically originates from the aromatic amino acids (72). The aim of the present work was to clarify the conformations of apoLDLj and apoLDL, by the following procedure: 1) measurement of the CD and ORD spectra of native LDLj and LDL,; 2) evaluation of the contributions of the carbohydrate and lipid moieties to the CD and ORD spectra; 3) determination of the CD and ORD spectra due to the protein moiety by subtracting the contributions of the lipid and carbohydrate moieties; 4) comparison of the corrected results for secondary structures of apoLDLi and apoLDL, in native LDLj and LDL, with those obtained in the presence of SDS; 5) determination of the pH- and temperature-dependent conformational changes; and 6) comparison of the results obtained from the CD spectra with those from fluorescence emission spectra. MATERIALS AND METHODS Materials—Total low density lipoprotein (LDL,
